The present invention relates generally to treatment of Kaposi""s sarcoma (xe2x80x9cKSxe2x80x9d), particularly AIDS-associated KS, with interleukin-12 (xe2x80x9cIL-12xe2x80x9d).
The reports in 1981 of Kaposi""s sarcoma (KS) associated with AIDS in homosexual males increased interest in this heretofore uncommon neoplastic disease. It soon became apparent that the KS associated with this epidemic was more fulminate than either the classical or the endemic African forms, generally following a rapidly progressive course. In the setting of AIDS, KS can progress to involve visceral organs and, particular pulmonary KS, is not infrequently a cause of death. Moreover, aggressive cutaneous and lymphatic involvement is often a cause of substantial morbidity. At one point, KS was the second most common AIDS-defining illness. However, as the epidemic has matured, and the definition of what constitutes AIDS has been modified, the incidence of KS as the initial AIDS-defining illness in patients has fallen. However, the absolute number of KS cases continues to rise and KS now frequently develops after patients have had another AIDS-defining illness. The distribution of KS in the setting of AIDS is not uniform among all groups at risk for HIV infection, with the majority of cases occurring in white, male homosexuals. In fact, KS is seven times more common in homosexual or bisexual men (27.3%) than in all other AIDS patients combined (3.9%).
There is no curative therapy for KS at present. While there is some evidence to suggest that antiretroviral therapy may, under certain circumstances, delay or even partially reverse the development of KS, this tumor generally requires specific therapy. Numerous modalities have been tried with various results. Interferon alpha has been useful in obtaining good responses particularly in patients with disease limited to the skin and withT4 cell counts that are above 200/mm3. However, this is not a cure, and interferons can have toxicities that often overlap with those of AZT and can interfere with antiretroviral therapy. At present, studies are ongoing to try to administer interferons with various antiretroviral therapies. Localized KS lesions are usually treated with radiation therapy. More aggressive or visceral lesions are generally treated with cytotoxic chemotherapy. Chemotherapy of KS with various agents has resulted in tumor responses that may be substantial; however, these responses tend to be incomplete, temporary, and are often short-lived. Also, many of these agents are associated with significant myelosuppression and immunosuppression, and patients often cannot tolerate therapy for long periods of time. In addition, treatment with many of these agents may predispose patients to the development of opportunistic infections (OI).
Although the pathogenesis of KS is not completely understood, there is a substantial body of evidence to show that the process of angiogenesis is central to the initiation and propagation of KS lesions. Kaposi""s sarcoma-derived cells cultured in vitro have been shown to secrete a variety of autocrine and paracrine growth factors, including some with potent angiogenic including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and interleukin-1 (IL-1). Many of these same factors, in addition to others such as scatter factor (SF) and HIV-1 Tat, have also been shown to stimulate in vitro growth of KS-derived spindle cells. These same KS cells, when inoculated subcutaneously into nude mice, were found to be able to induce the growth of a KS-like lesion that was of mouse tissue origin. In addition, similar lesions were induced by the co-inoculation of bFGF and Tat as well as Tat and heparin. Thus it would appear that KS-derived spindle cells secrete factors that, alone or in combination with Tat, are capable of inducing the formation of KS-like lesions. In addition, the growth and development of these lesions in mice can be inhibited by the systemic administration of agents or compounds with antiangiogenic activity such as tissue inhibitor of metalloproteinase-2 (TIMP-2) or tecogalan.
There are also data implicating endothelial cells as the cell of origin for the KS spindle cell. Cultured endothelial cells take on a spindle morphology when exposed to cytokines released in vitro by activated T lymphocytes, including many of the cytokines that induce in vitro KS-derived spindle cell proliferation. In addition, these endothelial cells also become sensitive to the in vitro proliferative effects of Tat in a manner similar to that of KS-derived spindle cells.
The initial stimulus or factor that initiates the proposed cytokine cascade leading to the development of KS is not known. However, there is a growing body of literature suggesting that a herpes-like virus, tentatively named Kaposi""s sarcoma herpes virus (KSHV) or human herpes virus-8 (HHV-8) may be involved. There are reports that this virus appears to be present in or associated with KS lesions from HIV-infected as well as classical and African KS at a much greater frequency than in patients without KS, although it has been reported in a number of other disease such as body cavity lymphomas and Castleman""s disease. In addition, there is a recent report that KSHV is present in the flat endothelial cells lining vascular spaces of KS lesions as well as in typical KS spindle cells. Thus, although the exact mechanisms and steps associated with the development of KS is not known, it is clear that angiogenesis is central to the overall pathogenesis of this disease.
Studies have shown that CD4-positive T-lymphocytes can be divided into two major groups: T helper type 1 cells (TH1) cells that produce interleukin-2 (IL-2) and interferon-gamma, and T helper type 2 cells (TH2) cells that mainly produce interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), and interleukin-10 (IL-10)1-4. In general, TH1 cells mediate cellular, type-1 immune responses while TH2 cells are involved in humoral type-2 immune responses. In the setting of HIV infection, there is a decrease in interleukin-2 production and other TH1-mediated type-1 reactions with an enhancement of TH2-mediated type-2 reactions, and this shift of immune responses away from type-1 predominance is felt to be a central feature of HIV infection5,6.
In summary, AIDS-associated KS is a serious neoplastic disorder associated with AIDS that can cause significant morbidity and even death. Treatment for this disorder at present is not optimal, and there is a definite need for newer agents that are better tolerated. As noted above, there is evidence that neovascularization is important in the development of KS. For this reason, it has been hypothesized that inhibition of angiogenesis may potentially benefit patients with KS. As will be described below, interleukin 12 (IL-12) has been shown to have potent antiangiogenic activity and is able to substantially stimulate TH1 immune responses.
Interleukin-12 (IL-12) is a cytokine that has been found to enhance the proliferation of activated T and natural killer (NK) cells; to enhance cytotoxic T and NK cell activity; and to induce interferon-gamma (IFN-xcex3) production7. Also, IL-12 has demonstrated anti-tumor and anti-metastatic activity in preclinical models8. In addition, IL-12 has been shown to potentiate the growth and differentiation of TH1 cells while inhibiting TH2 activity9. This effect of T helper differentiation by IL-12 has also been found to restore HIV-specific cell-mediated immune responses ex vivo in T cells from HIV-infected patients10.
More recently, Voest and colleagues have reported that IL-12 is a potent antiangiogenic agent11. They further found that the anti-angiogenic activity of IL-12 was mediated through induction of IFN-xcex3. Subsequently, Angiolillo and colleagues reported that human interferon-inducible protein 10 (IP-10), a chemokine induced by IFN-xcex3, is a potent inhibitor of in vivo angiogenesis12. Therefore, it appears that the antiangiogenic activity of IL-12 may be through its induction of local IFN-xcex3production with subsequent upregulation of IP-10 production. Thus, IL-12 has been shown to possess potent antiangiogenesis activity in vivo as well as to selectively enhance TH1 activity.
Foli and associates have recently reported the effects of IL-12 on in vitro HIV replication13. They showed that IL-12 induced in vitro replication of HIV in prestimulated, fresh peripheral blood mononuclear cells (PBMC) as determined by the production of HIV p24 antigen over 7 days of culture. This IL-12-induced HIV replication was not attributable to induction of IL-1, IL-2, tumor necrosis factor-alpha or -beta and was associated with a selective loss of the CD4 subset in stimulated cultures. However, IL-12 had little or no effect on HIV replication in monocyte/macrophages. Finally, they showed that the IL-12-induced increase in HIV replication could be inhibited by the dideoxy-nucleosides AZT, ddI, and ddC13. Thus, there is a possibility that systemically administered IL-12 may increase HIV replication in infected patients, particularly in the absence of antiretroviral therapy.
There have been numerous Phase I and Phase II trials administering IL-12 intravenously or subcutaneously employing a variety of doses and schedules. There were a number of toxicities noted. These included fever and constitutional symptoms; nausea, vomiting anorexia, diarrhea, stomatitis, dyspepsia, and guiac positive stools; transient decreases of leukocyte, lymphocyte, neutrophil, and platelet counts with anemia and occasional increases in PT and PTT; transient elevations of serum glucose values and less frequently hyperglycemia; dose-dependent elevations of SGOT, SGPT, alkaline phosphatase, and bilirubin; dyspnea; hematuria, proteinuria, elevated serum creatinine and blood urea nitrogen, and oliguria; confusion, anxiety, dizziness, insomnia, hypotonia, nervousness, somnolence, and tremor; hypotension and some peripheral edema; erythema and pruritis at the site of subcutaneous injections that resolved spontaneously; and increases in thyroid stimulating hormone (TSH).
Six patients died while receiving IL-12; 4 deaths were from disease progression while 2 were attributable to the IL-12. The two patients whose death was felt to be related to IL-12 were both enrolled on Phase II trials treating patients with renal cell carcinoma with 500 ng/kg of IL-12 intravenously dailyxc3x975 days every 3 weeks. One patient died on cycle 1 day 10 as the result of a lower gastrointestinal bleed, and the other died on cycle 1 day 23 due to multiple organ failure. It ultimately was determined that the toxicity profile of IL-12 was highly schedule dependent and that the dose administered in the Phase II trial was too high for that schedule. In particular, earlier Phase I studies had utilized a single dose of IL-12 with a delay before multiple doses were given, while the Phase II trial did not utilize this earlier single dose. There are data to suggest that through an as yet undetermined mechanism, this initial dose of IL-12 induced a sort of tolerance that reduced toxicity to subsequent doses.
There are data on single-dose, placebo-controlled Phase I trials administering IL-12 subcutaneously to HIV-infected patients with either 100-300 CD4 cells/mm3 or 301-500 CD4 cells/mm3. The dose levels studied were 3, 10, 30, 100, 300, and 1000 ng/kg. The maximally tolerated dose (MTD) was found to be 300 ng/kg on the basis of toxicity observed at the 1000 ng/kg dose (fatigue, stomatitis, aminotransferase elevations, and hyperbilirubinemia). There were no clinically significant changes in CD4 or CD8 cell counts or plasma viremia (measured by a branched-chain DNA assay) in any subject. There were, however, transient increases in interferon-gamma and neopterin levels seen in patients receiving doses greater than 100 ng/kg.
A Phase I/II double-blind, randomized, placebo-controlled, multicenter, dose-escalating study of IL-12 in HIV-infected patients having 100 to 500 CD4 cells/mm3 was initiated in November, 1995. Interleukin-12 is being administered by subcutaneous injection twice a week for 12 weeks. The dose levels to be studied are 30, 100, 300, and 500 ng/kg/injection of IL-12. Twenty-four patients will be enrolled in each dose cohorts (18 receiving IL-12 and 6 receiving placebo) to ensure that there will be 20 evaluable patients who complete at least 4 weeks of study drug administration. A total of 26 patients have been enrolled at the 30 ng/kg dose level. Four patients have been withdrawn from the study for non-safety-related reasons. Thirteen patients have completed 12 weeks of therapy. The 30 ng/kg dose has been well tolerated. To date, no serious adverse events related to study drug administration or dose-limiting toxicities have been reported. Dose escalation to the 100 ng/kg dose level has occurred and 10 patients have been enrolled at this dose level.
It would, therefore, be desirable to provide treatment for KS by administration of IL-12.
The present invention provides for a method for treating Kaposi""s sarcoma in a mammalian subject, which method comprises administering to the subject a therapeutically effective amountof IL-12 or a biologically active fragment or subunit thereof.
In other embodiments, the present invention provides for a method for inhibiting angiogenesis in a lesion associated with Kaposi""s sarcoma in a mammalian subject, which method comprising administering to the subject a therapeutically effective amount of IL-12 or a biologically active fragment or subunit thereof.
In preferred embodiments, the subject treated has AIDS. In other preferred embodiments, IL-12 is administered as a protein or in the form of DNA encoding IL-12.
In still other preferred embodiments, IL-12 protein is administered at a dose of from 100-1000 ng/kg of subject body weight, preferably at a dose selected from the group consisting of 300, 500, 625, and 750 ng/kg of subject body weight, and most preferably at a dose of 300 ng/kg of subject body weight.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.
Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor, is a heterodimeric cytokine described, for example, in M. Kobayashi et al., 1989, J. Exp. Med. 170: 827. IL-12 can be purified from natural sources, produced by chemical synthesis, or preferably produced by recombinant DNA techniques, for example by the expression and isolation of IL-12 protein in recombinant host cells as described in detail in International Patent Application WO90/05147, published May 17, 1990 (also European Patent Application No. 441,900), incorporated by reference herein. The DNA and amino acid sequences of the 30 kD and 40 kD subunits of the heterodimeric human IL-12 are provided in the above recited international application and in U.S. Pat. No. 5,571,515, incorporated by reference herein. Research quantities of recombinant human and murine IL-12 are also available from Genetics Institute, Inc., Cambridge, Mass.
As used herein, xe2x80x9cinterleukin-12xe2x80x9d and xe2x80x9cIL-12xe2x80x9d refer to interleukin-12, its individual subunits, fragments thereof which exhibit IL-12 adjuvant activity, polynucleotides encoding IL-12, and functional equivalents of xe2x80x9cinterleukin-12xe2x80x9d and xe2x80x9cIL-12xe2x80x9d.
A therapeutically effective amount of IL-12 is an amount that when administered results in (1) diminution of KS symptoms, including without limitation a decrease in the number, recurrence, spread, or size of one or more KS lesions, or (2) a reduction in angiogenesis in one or more existing KS lesions or in the formation of new KS lesions. The amount of IL-12 administered to the host will vary depending on a variety of other factors, including the antigen(s) employed, the size, age, body weight, general health, sex, and diet of the host, the time or duration of administration, and the particular qualities of the KS lesions being treated. As one example, a therapeutically effective amount of IL-12 polypeptide is desirably between about 10 ng to about 1000 ng (preferably about 100 ng to about 750 ng, or about 100 ng to about 300 ng, or about 300 ng to about 500 ng, or about 500 ng to about 750 ng) of IL-12 polypeptide per kg patient body weight. Preferred doses are 100 ng/kg, 300 ng/kg, 500 ng/kg, 625 ng/kg, and 750 ng/kg. The effective amount for any particular patient will be readily defined by balancing the efficacy and toxicity of the IL-12 administration. Adjustment and manipulation of established dose ranges are well within the ability of those skilled in the art.
The IL-12 can be administered to a host in a variety of ways. The routes of administration include without limitation intradermal, transdermal (for example, by slow-release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, aural, epidural, anal or vaginal (for example, by suppositories), and intranasal routes. Any other convenient route of administration can be used, for example, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings. Particularly prefered routes of administration are those which patients can conveniently administer to themselves in in-home settings, such as without limitation intradermal, transdermal (for example, by slow-release polymers), intravenous, subcutaneous, oral, aural, epidural, anal or vaginal (for example, by suppositories), or intranasal routes of administration, or absorption through epithelial or mucocutaneous linings. In addition, the IL-12 can be administered in combination with other components or biologically active agents, such as any of a variety of anti-viral agents (for example, nucleoside analog reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors) or any of a variety of anti-neoplastic or chemotherapeutic agents (for example, acyclic nucleoside phosphonates, Adriamycin, bleomycin, and/or vincristine, either individually or in combination), or other biologically active agents such as tretinoin. Other pharmaceutically acceptable components may also be administered in combination with IL-12, for example surfactants such as glycerides, excipients such as lactose, carriers diluents, and vehicles. If desired, certain sweetening, flavoring, and/or coloring agents can also be added.
Further, IL-12 can be administered by in vivo expression in the host of polynucleotides encoding IL-12. The polynucleotides, preferably in the form of DNA, may be delivered to the patient for in vivo expression of IL-12. So-called xe2x80x98naked DNAxe2x80x99 may be used to express IL-12 in vivo in a host. (Cohen, J., 1993, Science 259: 1691-1692; Fynan, E. et al., 1993, PNAS USA 90: 11478-11482; and Wolff, J. A. et al., 1991, Biotechniques 11:474-485 describe similar uses of xe2x80x98naked DNAxe2x80x99, all incorporated by reference herein.)
Still other modes of delivering IL-12 to the patient in the form of polynucleotides encoding them are known to those of skill in the art and may be employed rather than administration of IL-12 polypeptides, as desired. For example, polynucleotides encoding IL-12 may be administered as part of a vector or as a cassette containing the sequences encoding IL-12 operatively linked to a promoter sequence. (For example, see International Patent Application PCT WO94/01139, published Jan. 20, 1994 and incorporated by reference herein.) Briefly, the DNA encoding IL-12 protein or desired fragments thereof may be inserted into a nucleic acid cassette. This cassette may be engineered to contain, in addition to the IL-12 sequence to be expressed, other optional flanking sequences which enable its insertion into a vector. This cassette may then be inserted into an appropriate vector downstream of a promoter, an mRNA leader sequence, an initiation site, and other regulatory sequences capable of directing the replication and expression of that sequence in vivo. Additional regulatory sequences may be inserted downstream of the coding sequence to be expressed. This vector permits in vivo expression of the IL-12 polypeptides within the host.
Patent and literature references cited herein are incorporated by reference as if fully set forth.